Methods of administering and evaluating nitrogen scavenging drugs for the treatment of hepatic encephalopathy

ABSTRACT

The present disclosure provides methods for treating hepatic encephalopathy (HE) and for optimizing and adjusting nitrogen scavenging drug dosage for subjects with HE.

RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationNo. 61/728,967, filed Nov. 21, 2012, and U.S. Provisional ApplicationNo. 61/759,292, filed Jan. 31, 2013, the disclosures of which areincorporated by reference herein in their entirety, including drawings.

BACKGROUND

Hepatic encephalopathy (HE) refers to a spectrum of neurologic signs andsymptoms believed to result from increased blood ammonia levels, whichfrequently occur in subjects with cirrhosis or certain other types ofliver disease. Subjects with HE typically show altered mental statusranging from subtle changes to coma, features similar to those insubjects with urea cycle disorders (UCDs).

Glycerol phenylbutyrate (glyceryl tri-[4-phenylbutyrate]) (HPN-100, GPB,GT4P, glycerol PBA), which is described in U.S. Pat. No. 5,968,979, iscurrently under development for treatment of HE. Like sodium PBA (NaPBA,approved in the United States as BUPHENYL® and in Europe as AMMONAPS®)and sodium benzoate, HPN-100 is a nitrogen scavenging agent. These drugsare often referred to as alternate pathway drugs because they providethe body with an alternate pathway to urea for excretion of wastenitrogen (Brusilow 1980; Brusilow 1991).

NaPBA is a phenylacetic acid (PAA) prodrug, while HPN-100 is a prodrugof PBA and a pre-prodrug of PAA. HPN-100 and NaPBA share the samegeneral mechanism of action: PBA is converted to PAA via beta oxidation,and PAA is conjugated enzymatically with glutamine to formphenylacetylglutamine (PAGN), which is excreted in the urine. Thestructures of PBA, PAA, and PAGN are set forth below.

The clinical benefit of NaPBA and HPN-100 with regard to nitrogenretention disorders such as HE derives from the ability of PAGN toeffectively replace urea as a vehicle for waste nitrogen excretionand/or to reduce the need for urea synthesis (Brusilow 1991; Brusilow1993). Because each glutamine contains two molecules of nitrogen, thebody rids itself of two waste nitrogen atoms for every molecule of PAGNexcreted in the urine (FIG. 1). Therefore, two equivalents of nitrogenare removed for each mole of PAA converted to PAGN. PAGN represents thepredominant terminal metabolite, and one that is stoichiometricallyrelated to waste nitrogen removal, a measure of efficacy in the case ofnitrogen retention states. The difference between HPN-100 and NaPBA withrespect to metabolism is that HPN-100 is a triglyceride and requiresdigestion, presumably by pancreatic lipases, to release PBA (McGuire2010).

In contrast to NaPBA or HPN-100, sodium benzoate acts when benzoic acidis combined enzymatically with glycine to form hippuric acid. For eachmolecule of hippuric acid excreted in the urine, the body rids itself ofone waste nitrogen atom.

Methods of determining an effective dosage of PAA prodrugs such as NaPBAor HPN-100 for a subject in need of treatment for a nitrogen retentiondisorder are described in WO09/1134460 and WO10/025303. While ammoniahas long been suspected as important in the pathogenesis of HE, the dataare largely correlative and it is only recently that an ammoniaselective intervention has been shown to reduce the likelihood of HEevents (Rockey 2012). Ammonia is not routinely monitored in patientswith HE; rather, treatment is based on clinical assessment.

SUMMARY

Provided herein in certain embodiments are methods of treating hepaticencephalopathy in a subject in need thereof by administering a nitrogenscavenging drug at a dosage sufficient to maintain a fasting bloodammonia level at or below a specified threshold level with respect tothe upper limit of normal for blood ammonia. In certain of theseembodiments, the threshold level is 1.5 times the upper limit of normalfor blood ammonia, and in certain embodiments the upper limit of normalfor blood ammonia is 35 μmol/L. In certain embodiments, the nitrogenscavenging drug is HPN-100, PBA, NaPBA, sodium benzoate, or anycombination thereof (i.e., any combination of two or more of HPN-100,PBA, NaPBA).

Provided herein in certain embodiments are methods of treating hepaticencephalopathy in a subject in need thereof by administering a firstdosage of a nitrogen scavenging drug, measuring fasting blood ammonialevel, and comparing the fasting blood ammonia level to the upper limitof normal for blood ammonia to determine whether to increase the dosageof the drug, wherein the dosage needs to be increased if the fastingblood ammonia level is at or above a specified threshold level withrespect to the upper limit of normal. In certain of these embodiments,the threshold level is 1.5 times the upper limit of normal for bloodammonia, and in certain embodiments the upper limit of normal for bloodammonia is 35 μmol/L. In certain embodiments, these methods include anadditional step of administering a second dosage of the drug based onthe comparison of fasting blood ammonia level to the upper limit ofnormal for blood ammonia. In certain embodiments, the nitrogenscavenging drug is HPN-100, PBA, NaPBA, sodium benzoate, or anycombination thereof (i.e., any combination of two or more of HPN-100,PBA, NaPBA).

Provided herein in certain embodiments are methods of treating hepaticencephalopathy in a subject in need thereof by measuring fasting bloodammonia level, comparing the fasting blood ammonia level to the upperlimit of normal for blood ammonia, and administering a nitrogenscavenging drug if the fasting blood ammonia level is at or above aspecified threshold level with respect to the upper limit of normal. Incertain of these embodiments, the threshold level is 1.5 times the upperlimit of normal for blood ammonia, and in certain embodiments the upperlimit of normal for blood ammonia is 35 μmol/L. In certain embodiments,the nitrogen scavenging drug is HPN-100, PBA, NaPBA, sodium benzoate, orany combination thereof (i.e., any combination of two or more ofHPN-100, PBA, NaPBA). In certain embodiments, the subject has previouslyreceived a first dosage of a nitrogen scavenging drug, and in certain ofthese embodiments the nitrogen scavenging drug is administered at adosage greater than the first dosage if the fasting blood ammonia levelis at or above the specified threshold level with respect to the upperlimit of normal.

Provided herein in certain embodiments are methods of treating hepaticencephalopathy in a subject in need thereof who has previously beenadministered a first dosage of a nitrogen scavenging drug by measuringfasting blood ammonia level, comparing the fasting blood ammonia levelto the upper limit of normal for blood ammonia, and administering asecond dosage of the drug that is greater than the first dosage if thefasting blood ammonia level is at or above a specified threshold levelwith respect to the upper limit of normal. In certain of theseembodiments, the threshold level is 1.5 times the upper limit of normalfor blood ammonia, and in certain embodiments the upper limit of normalfor blood ammonia is 35 μmol/L. In certain embodiments, the nitrogenscavenging drug is HPN-100, PBA, NaPBA, sodium benzoate, or anycombination thereof (i.e., any combination of two or more of HPN-100,PBA, NaPBA).

Provided herein in certain embodiments are methods of optimallyadministering a nitrogen scavenging drug for the treatment of hepaticencephalopathy in a subject in need thereof that include a step ofadministering the nitrogen scavenging drug, wherein the dosage of thedrug is adjusted to maintain a fasting blood ammonia at or below aspecified threshold level with respect to the upper limit of normal forblood ammonia. In certain of these embodiments, the threshold level is1.5 times the upper limit of normal for blood ammonia, and in certainembodiments the upper limit of normal for blood ammonia is 35 μmol/L. Incertain embodiments, the nitrogen scavenging drug is HPN-100, PBA,NaPBA, sodium benzoate, or any combination thereof (i.e., anycombination of two or more of HPN-100, PBA, NaPBA).

Provided herein in certain embodiments are methods of optimizing thedosage of a nitrogen scavenging drug for the treatment of hepaticencephalopathy in a subject in need thereof by administering a firstdosage of a nitrogen scavenging drug, measuring fasting blood ammonialevel, and comparing the fasting blood ammonia level to the upper limitof normal for blood ammonia to determine whether to increase the dosageof the drug, wherein the dosage needs to be increased if the fastingblood ammonia level is at or above a specified threshold level withrespect to the upper limit of normal. In certain of these embodiments,the threshold level is 1.5 times the upper limit of normal for bloodammonia, and in certain embodiments the upper limit of normal for bloodammonia is 35 μmol/L. In certain embodiments, these methods include anadditional step of administering a second dosage of the drug based onthe comparison of fasting blood ammonia level to the upper limit ofnormal for blood ammonia. In certain embodiments, the nitrogenscavenging drug is HPN-100, PBA, NaPBA, sodium benzoate, or anycombination thereof (i.e., any combination of two or more of HPN-100,PBA, NaPBA).

Provided herein in certain embodiments are methods of adjusting thedosage of a nitrogen scavenging drug for the treatment of hepaticencephalopathy in a subject in need thereof by administering a firstdosage of a nitrogen scavenging drug, measuring fasting blood ammonialevel, and determining whether the drug dosage needs to be adjustedbased on the fasting blood ammonia level, wherein a fasting bloodammonia level at or above a specified threshold level with respect tothe upper limit of normal for blood ammonia indicates that the dosageneeds to be increased. In certain of these embodiments, the thresholdlevel is 1.5 times the upper limit of normal for blood ammonia, and incertain embodiments the upper limit of normal for blood ammonia is 35μmol/L. In certain embodiments, these methods include an additional stepof administering a second dosage of the drug based on the comparison offasting blood ammonia level to the upper limit of normal for bloodammonia. In certain embodiments, the nitrogen scavenging drug isHPN-100, PBA, NaPBA, sodium benzoate, or any combination thereof (i.e.,any combination of two or more of HPN-100, PBA, NaPBA).

Provided herein in certain embodiments are methods of treating hepaticencephalopathy in a subject in need thereof by determining a targeturinary phenylacetyl glutamine output, calculating an effective initialdosage of a PAA prodrug to achieve the target urinary phenylacetylglutamine output based on a mean conversion of PAA prodrug to urinaryphenylacetyl glutamine of 52% to 63%, and administering the effectiveinitial dosage of PAA prodrug to the subject. In certain embodiments,the PAA prodrug is HPN-100, PBA, or NaPBA.

Provided herein in certain embodiments are methods of administering aPAA prodrug for the treatment of hepatic encephalopathy in a subject inneed thereof by administering a first dosage of the PAA prodrug,determining urinary phenylacetyl glutamine output followingadministration of the first dosage, determining an effective dosage ofthe PAA prodrug based on the urinary phenylacetyl glutamine output,wherein the effective dosage is based on a mean conversion of PAAprodrug to urinary PAGN of 52% to 63%, and administering the effectivedosage to the subject.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: The urea cycle and how certain nitrogen-scavenging drugs mayassist in elimination of excessive ammonia.

FIG. 2: Estimated number of HE events per subject over 16 weeks versuspre-dose plasma ammonia level on day 1 (baseline). Minimum and maximumobserved plotted for reference. Levels 4 and 5 differ significantly fromlevel 1 (p=0.007 and p=0.002, respectively).

FIG. 3: Estimated number of HE events per subject over 16 weeks versuspre-dose plasma ammonia level on day 7. Minimum and maximum observedplotted for reference. Levels 4 and 5 differ significantly from level 1(p=0.0009 and p=0.0003, respectively).

FIG. 4: Estimated number of HE events per subject over 16 weeks versusplasma ammonia level on day 7, four hours after dosing. Minimum andmaximum observed plotted for reference. Levels 4 and 5 differsignificantly from level 1 (p=0.002 and p=0.001, respectively).

FIG. 5: Estimated number of HE events per subject over 16 weeks versuspre-dose plasma ammonia level on day 14. Minimum and maximum observedplotted for reference. Levels 4 and 5 differ significantly from level 1(p=0.014 and p=0.001, respectively).

FIG. 6: Estimated number of HE events per subject over 16 weeks versusplasma ammonia level on day 14, four hours after dosing. Minimum andmaximum observed plotted for reference. Level differs significantly fromlevel 1 (p=0.002).

FIG. 7: Probability of having 0, 1, or 2 or more HE events over 16 weeksas predicted by plasma ammonia levels at baseline (day 1, pre-dose).

FIG. 8: Probability of having 0 or 1 or more HE events over 16 weeks aspredicted by plasma ammonia levels at baseline (day 1, pre-dose).

FIG. 9: Probability of having 0, 1, or 2 or more HE events over 16 weeksas predicted by plasma ammonia levels (day 7, pre-dose).

FIG. 10: Probability of having 0 or 1 or more HE events over 16 weeks aspredicted by plasma ammonia levels (day 7, pre-dose).

DETAILED DESCRIPTION

The following description of the invention is merely intended toillustrate various embodiments of the invention. As such, the specificmodifications discussed are not to be construed as limitations on thescope of the invention. It will be apparent to one skilled in the artthat various equivalents, changes, and modifications may be made withoutdeparting from the scope of the invention, and it is understood thatsuch equivalent embodiments are to be included herein.

In subjects with HE, the desired effect of treatment with a nitrogenscavenging drug is prevention of HE events or reduction of the frequencyof HE events and their associated cost and morbidity. The assessment oftreatment effect and interpretation of ammonia levels is confounded bythe fact that individual ammonia values vary several-fold over thecourse of a day and are impacted by timing of the blood draw in relationto the last meal and dose of drug (see, e.g., Lee 2010; Lichter-Konecki2011; Diaz 2011, Ghabril 2012).

A random ammonia value obtained during an outpatient visit may fail toprovide a reliable measure of a subject's status and the drug effect.For example, basing treatment on a blood sample taken after eating ameal might overestimate average daily ammonia level and result inovertreatment. Conversely, basing treatment on a blood sample takenafter drug administration might underestimate average daily ammonialevel and result in undertreatment. Furthermore, subjects may notexhibit dramatically increased ammonia levels between HE events, andassessment of ammonia level at the time of an HE event is not alwayspossible. A more accurate view of daily ammonia level could be obtainedby multiple blood draws in a controlled setting over an extended periodof time. Although this is currently done in clinical trials, it isclinically impractical.

As set forth in the examples section below, the relationship betweenfasting ammonia levels and the likelihood of patients experiencing an HEevent was evaluated in subjects with cirrhosis and HE. It was found thatincreased fasting ammonia levels correlated strongly with the likelihoodof experiencing an HE event. Surprisingly, the relationship betweenammonia levels and HE events was non-linear, and there was a step up inHE event risk at a fasting ammonia level of around 1.5 times the upperlimit of normal (ULN) rather than at the ULN. Based on these results, acomparison of fasting ammonia levels to a specified threshold or targetrange with respect to the ULN for blood ammonia represents a novel,clinically useful, and practical predictor of HE event risk. The presentapplication provides practical applications of this finding in the formof methods and kits for treating HE, optimally administering a nitrogenscavenging drug for the treatment of HE, adjusting and optimizing thedosage of a nitrogen scavenging drug for the treatment of HE, evaluatingthe efficacy of a nitrogen scavenging drug for the treatment of HE,determining whether to administer a nitrogen scavenging drug for thetreatment of HE, predicting the likelihood or risk of an HE event,evaluating and monitoring ammonia exposure, and other relatedembodiments.

Provided herein are threshold levels and target ranges for fasting bloodammonia upon which an effective dosage of a nitrogen scavenging drug forthe treatment of HE can be based. An effective dosage of a nitrogenscavenging drug as used herein refers to a dosage that results in afasting blood ammonia level falling at or below a specified thresholdlevel or within a specified target range after one or moreadministrations. In certain embodiments, the effective dosage results ina fasting blood ammonia level falling at or below a specified thresholdlevel or within a specified target range after multiple administrations,and in certain of these embodiments the effective dosage results in afasting blood ammonia level falling at or below a specified thresholdlevel or within a specified target range after the drug has reachedsteady state. In certain embodiments, steady state for a particulardosage of a nitrogen scavenging drug is reached at around three daysafter the initial administration of that dosage. In other embodiments,steady state may be reached at two, four, or five days after the initialadministration.

Threshold levels and target ranges for fasting blood ammonia are basedon the ULN for blood ammonia. In certain embodiments, a specified targetrange for fasting blood ammonia is <1.2, ≦1.2, <1.3, ≦1.3, <1.4, ≦1.4,<1.5, ≦1.5, <1.6, ≦1.6, <1.7, ≦1.7, <1.8, ≦1.8, <1.9, ≦1.9, <2.0, or≦2.0 times the ULN for blood ammonia. In certain of these embodiments,the specified target range is <1.5 or ≦1.5 times the ULN for bloodammonia. In other embodiments, a specified target range for fastingblood ammonia is 0.1 to 1.5, 0.5 to 1.5, 0.7 to 1.3, 0.8 to 1.2, or 1 to1.5 times the ULN for blood ammonia. In certain embodiments, a specifiedthreshold level for fasting blood ammonia is 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, or 2.0 times the ULN for blood ammonia. In certain ofthese embodiments, the specified threshold level is 1.5 times the ULNfor blood ammonia. In certain embodiments, a fasting blood ammonia levelthat is at or above the specified threshold level or above the specifiedtarget range indicates that a subject needs to be administered anitrogen scavenging drug or, where the subject has received a nitrogenscavenging drug previously, that the subject needs to be administered adifferent nitrogen scavenging drug or a higher dosage of the originalnitrogen scavenging drug. Similarly, in certain embodiments a fastingblood ammonia level that is at or below the specified threshold level orwithin the specified target range indicates that the subject does notneed to be administered a nitrogen scavenging drug or, where the subjecthas received a nitrogen scavenging drug previously, that the subjectshould continue to be administered the same nitrogen scavenging drugand/or the same dosage. In certain embodiments, the optimal range forfasting blood ammonia includes the specified threshold level. In theseembodiments, a fasting blood ammonia level at or below the specifiedthreshold level is considered acceptable or optimal. For example, wherethe specified threshold level is 1.5, nitrogen scavenging drugadministration may be started or increased if the fasting blood ammonialevel is above 1.5. In other embodiments, the optimal range for fastingblood ammonia does not include the specified threshold level. In theseembodiments, only a fasting blood ammonia level below the specifiedthreshold level is considered acceptable or optimal. For example, wherethe specified threshold level is 1.5, nitrogen scavenging drugadministration may be started or increased if a subject exhibits afasting blood ammonia level at or above 1.5. An effective dosage of anitrogen scavenging drug may be an initial dosage,subsequent/maintenance dosage, improved dosage, or a dosage determinedin combination with other factors. In certain embodiments, the effectivedosage may be the same as or different than an initial dosage. In otherembodiments, the effective dosage may be higher or lower than an initialdosage.

Provided herein in certain embodiments are methods of treating HE in asubject in need thereof. The terms “treat,” “treating,” or “treatment”as used herein with regard to HE may refer to preventing HE events,reducing the number or frequency of HE events, decreasing the likelihoodof experiencing an HE event, preventing, delaying, reducing, or endingsymptoms associated with HE or HE events, or some combination thereof.

Overt HE events are often defined in the art as West Haven grade ≧2,typically manifested by confusion to time, place, person and/orsomnolence and even coma. In addition, a West Haven grade 1 and anincrease in asterixis scales for patients who are West Haven grade 0when in remission is considered clinically significant. As used herein,the “HE event” refers to a West Haven Grade 2 or above or (2) a WestHaven Grade 1 AND an asterixis grade increase of 1, if the baseline WestHaven was 0.

A “subject in need thereof” as used herein refers to a human subjecthaving HE, suspected of having HE, deemed at risk for developing HEbased on one or more genetic or environmental factors, currently orpreviously experiencing an HE event, or deemed at risk of experiencing afuture HE event.

In certain embodiments, the methods of treating HE provided hereincomprise administering a nitrogen scavenging drug at a dosage sufficientto drop a subject's fasting blood ammonia level to or below a specifiedthreshold level or to within a specified target range with respect tothe ULN for blood ammonia, or to maintain the subject's fasting bloodammonia level at or below the specified threshold level or within thespecified target range for a specific period of time (e.g., 2 days, 4days, 1 week, 1 month, or indefinitely). For example, the drug may beadministered at a dosage sufficient to maintain a subject's fastingblood ammonia level at or below a specified threshold level of 1.5 timesthe ULN, or at a dosage sufficient to maintain the subject's fastingblood ammonia level within a specified target range of 1 to 1.5, between1 and 1.5, <1.5, or ≦1.5 times the ULN. In certain embodiments,administration of the nitrogen scavenging drug at a dosage sufficient tomaintain a fasting blood ammonia level at or below a specified thresholdlevel or within a specified target range with respect to the ULN forblood ammonia decreases the likelihood of the subject experiencing an HEevent.

In certain embodiments, the methods of treating HE provided hereincomprise (a) measuring a fasting blood ammonia level, (b) comparing thefasting blood ammonia level to the ULN for blood ammonia to determinewhether to administer a nitrogen scavenging drug, and (c) administeringa nitrogen scavenging drug if the fasting blood ammonia level is at orabove a specified threshold level or above a specified target range withrespect to the ULN. In certain embodiments, the specified thresholdlevel is 1.5 times the ULN. In certain embodiments, the specified targetrange is 1 to 1.5, between 1 and 1.5, <1.5, or ≦1.5 times the ULN. Incertain embodiments, administration of the nitrogen scavenging drugdecreases the likelihood of the subject experiencing an HE event. Incertain embodiments, the steps are repeated until a fasting bloodammonia level at or below the threshold level or within the target rangeis reached or maintained. In those embodiments where the steps arerepeated, subsequent dosages may be the same as or different than thefirst dosage. For example, a second dosage may be administered that isgreater than the first dosage if the first dosage was insufficient tolower fasting blood ammonia level to at or below the threshold level orto within the target range.

In certain embodiments, the methods of treating HE provided hereincomprise (a) administering a first dosage of a nitrogen scavenging drug,(b) measuring a fasting blood ammonia level, and (c) comparing thefasting blood ammonia level to the ULN for blood ammonia to determinewhether to increase the dosage of the nitrogen scavenging drug. In theseembodiments, the dosage needs to be increased if the fasting bloodammonia level is at or above a specified threshold level or above aspecified target range with respect to the ULN. In certain of theseembodiments, these methods include an additional step of administering asecond dosage of the drug greater than the first dosage based on thecomparison in step (c). If the fasting blood ammonia level is at orbelow the specified threshold level or within the specified targetrange, on the other hand, the second dosage may be the same as or lessthan the first dosage. In certain embodiments, these steps may berepeated, with the subject receiving increasing dosages of nitrogenscavenging drug until a fasting blood ammonia level at or below thespecified threshold or within target range is reached or maintained. Forexample, in certain embodiments fasting blood ammonia level may bemeasured after administration of the second dosage, and if the fastingblood ammonia level is at or above the specified threshold level orabove the target range with respect to the ULN, a third dosage may beadministered that is greater than the second dosage. In certainembodiments, the specified threshold level is 1.5 times the ULN. Incertain embodiments, the specified target range is 1 to 1.5, between 1and 1.5, <1.5, or ≦1.5 times the ULN. In certain embodiments,administration of the second, third, or subsequent dosage of the drugdecreases the likelihood of the subject experiencing an HE event.

In certain embodiments, the methods of treating HE provided herein aredirected to treatment of subjects who have previously received a firstdosage of a nitrogen scavenging drug. In certain of these embodiments,the methods comprise (a) measuring a fasting blood ammonia level, (b)comparing the fasting blood ammonia level to the ULN for blood ammonia,and (c) administering a second dosage of the drug that is greater thanthe first dosage if the fasting blood ammonia level is at or above aspecified threshold level or above a target range with respect to theULN. If the fasting blood ammonia level is at or below the specifiedthreshold level or within the specified target range, on the other hand,the second dosage may be the same as or less than the first dosage. Incertain embodiments, these steps may be repeated. For example, incertain embodiments fasting blood ammonia level may be measured afteradministration of the second dosage, and if the fasting blood ammonialevel is at or above the specified threshold level or above the targetrange with respect to the ULN, a third dosage may be administered thatis greater than the second dosage. This process may be repeated untilthe subject exhibits a fasting ammonia level at or below the specifiedthreshold level or within the specified target range. In certainembodiments, the specified threshold level is 1.5 times the ULN. Incertain embodiments, the specified target range is 1 to 1.5, between 1and 1.5, <1.5, or ≦1.5 times the ULN. In certain embodiments,administration of the second, third, or subsequent dosage of thenitrogen scavenging drug decreases the likelihood of the subjectexperiencing an HE event.

Provided herein in certain embodiments are methods of optimallyadministering a nitrogen scavenging drug to a subject in need thereoffor treatment of HE. In certain embodiments, these methods compriseadministering the nitrogen scavenging drug at a dosage sufficient tolower a subject's fasting blood ammonia to or below a specifiedthreshold level or to within a specified target range with respect tothe ULN for blood ammonia, or to maintain the subject's fasting bloodammonia level at or below the threshold level or within the targetrange. For example, in certain embodiments the drug may be administeredat a dosage sufficient to maintain a subject's fasting blood ammonialevel below a specified threshold level of 1.5 times the ULN, or withina specified target range of 1 to 1.5, between 1 and 1.5, <1.5, or ≦1.5times the ULN. In certain embodiments, administration of the nitrogenscavenging drug decreases the likelihood of the subject experiencing anHE event.

In certain embodiments, the methods of optimally administering anitrogen scavenging drug for the treatment of HE provided hereincomprise (a) measuring a fasting blood ammonia level, (b) comparing thefasting blood ammonia level to the ULN for blood ammonia to determinewhether to administer a nitrogen scavenging drug, and (c) administeringa first dosage of a nitrogen scavenging drug if the fasting bloodammonia level is at or above a specified threshold level or above aspecified target range with respect to the ULN. In certain embodiments,the specified threshold level is 1.5 times the ULN. In certainembodiments, the specified target range is 1 to 1.5, between 1 and 1.5,<1.5, or ≦1.5 times the ULN. In certain embodiments, the steps arerepeated until a fasting blood ammonia level at or below the thresholdlevel or within the target range is reached or maintained. In thoseembodiments where the steps are repeated, the dosage of the nitrogenscavenging drug may be adjusted with each subsequent administration inorder to obtain a fasting blood ammonia level at or below the thresholdlevel or within the target range.

In certain embodiments, the methods of optimally administering anitrogen scavenging drug for the treatment of HE provided hereincomprise (a) administering a first dosage of a nitrogen scavenging drug,(b) measuring a fasting blood ammonia level, and (c) comparing thefasting blood ammonia level to the ULN for blood ammonia to determinewhether to increase the dosage of the drug, wherein the dosage needs tobe increased if the fasting blood ammonia level is at or above aspecified threshold level or above a specified target range with respectto the ULN. In certain embodiments, these methods include an additionalstep of administering a second dosage of the drug based on thecomparison step (c). If the fasting blood ammonia level is at or belowthe specified threshold level or within the specified target range, onthe other hand, the second dosage may be the same as or less than thefirst dosage. In certain embodiments, these steps may be repeated, withthe subject receiving increasing dosages of nitrogen scavenging druguntil a fasting blood ammonia level at or below the specified thresholdor within the specified target range is reached or maintained. Forexample, in certain embodiments fasting blood ammonia level may bemeasured after administration of the second dosage, and if the fastingblood ammonia level is at or above the specified threshold level orabove the specified target range with respect to the ULN, a third dosagemay be administered that is greater than the second dosage. In certainembodiments, the specified threshold level is 1.5 times the ULN. Incertain embodiments, the specified target range is 1 to 1.5, between 1and 1.5, <1.5, or ≦1.5 times the ULN. In certain embodiments,administration of the nitrogen scavenging drug decreases the likelihoodof the subject experiencing an HE event.

In certain embodiments, the methods of optimally administering anitrogen scavenging drug for the treatment of HE provided herein aredirected to subjects who have previously received a first dosage of anitrogen scavenging drug. In certain of these embodiments, the methodscomprise (a) measuring a fasting blood ammonia level, (b) comparing thefasting blood ammonia level to the ULN for blood ammonia, and (c)administering a second dosage of the drug that is greater than the firstdosage if the fasting blood ammonia level is at or above a specifiedthreshold level or above a specified target range with respect to theULN. If the fasting blood ammonia level is at or below the specifiedthreshold level or within the specified target range, on the other hand,the second dosage may be the same as or less than the first dosage. Incertain embodiments, these steps may be repeated. For example, incertain embodiments fasting blood ammonia level may be measured afteradministration of the second dosage, and if the fasting blood ammonialevel is at or above the specified threshold level or above thespecified target range with respect to the ULN, a third dosage may beadministered that is greater than the second dosage. This process may berepeated until the subject exhibits a fasting ammonia level at or belowthe specified threshold level or within the specified target range. Incertain embodiments, the specified threshold level is 1.5 times the ULN.In certain embodiments, the specified target range is 1 to 1.5, between1 and 1.5, <1.5, or ≦1.5 times the ULN. In certain embodiments,administration of the second, third, or subsequent dosage of thenitrogen scavenging drug decreases the likelihood of the subjectexperiencing an HE event.

Provided herein in certain embodiments are methods of adjusting thedosage of a nitrogen scavenging drug for the treatment of HE in asubject in need thereof. In certain embodiments, these methods compriseadjusting the dosage of a nitrogen scavenging drug to lower a subject'sfasting blood ammonia to or below a specified threshold level or towithin a specified target range with respect to the ULN for bloodammonia, or to maintain the subject's fasting blood ammonia level at orbelow the threshold level or within the specified target range. Forexample, the dosage may be adjusted to a dosage sufficient to maintain asubject's fasting blood ammonia level at or below a specified thresholdlevel of 1.5 times the ULN or within a specified target range of 1 to1.5, between 1 and 1.5, <1.5, or ≦1.5 times the ULN. In certainembodiments, administration of the adjusted dosage of nitrogenscavenging drug decreases the likelihood of the subject experiencing anHE event.

In certain embodiments, the methods of adjusting the dosage of anitrogen scavenging drug for the treatment of HE provided hereincomprise (a) administering a first dosage of a nitrogen scavenging drug,(b) measuring a fasting blood ammonia level, and (c) determining whetherthe drug dosage needs to be adjusted based on the fasting blood ammonialevel, wherein a fasting blood ammonia level at or above a specifiedthreshold level or above a specified target range with respect to theULN for blood ammonia indicates that the dosage needs to be increased.In certain embodiments, these methods include an additional step ofadministering an adjusted second dosage of the drug based on thecomparison step (c). In certain embodiments, if the fasting bloodammonia level in step (b) is at or below the specified threshold levelor within the specified target range, the adjusted second dosage may bethe same as or less than the first dosage. In certain embodiments, thesteps may be repeated, with the subject receiving increasing dosages ofnitrogen scavenging drug until a fasting blood ammonia level at or belowthe specified threshold level or within the specified target range isreached or maintained. For example, in certain embodiments fasting bloodammonia level may be measured after administration of the second dosage,and if the fasting blood ammonia level is at or above the specifiedthreshold level or above the specified target range with respect to theULN, a third dosage may be administered that is greater than the seconddosage. In certain embodiments, the specified threshold level is 1.5times the ULN. In certain embodiments, the specified target range is 1to 1.5, between 1 and 1.5, <1.5, or ≦1.5 times the ULN. In certainembodiments, administration of the nitrogen scavenging drug decreasesthe likelihood of the subject experiencing an HE event.

In certain embodiments, the methods of adjusting the dosage of anitrogen scavenging drug for the treatment of HE provided herein aredirected to subjects who have previously received a first dosage of anitrogen scavenging drug. In certain of these embodiments, the methodscomprise (a) measuring a fasting blood ammonia level and (b) determiningwhether the drug dosage needs to be adjusted based on the fasting bloodammonia level, wherein a fasting blood ammonia level at or above aspecified threshold level or above a specified target range with respectto the ULN for blood ammonia indicates that the dosage needs to beincreased. In certain embodiments, these methods include an additionalstep of administering an adjusted second dosage of the drug based on thecomparison step (c). In certain embodiments, if the fasting bloodammonia level in step (a) is at or below the specified threshold levelor within the specified target range, the adjusted second dosage may bethe same as or less than the first dosage. In certain embodiments, thesteps may be repeated, with the subject receiving increasing dosages ofnitrogen scavenging drug until a fasting blood ammonia level at or belowthe specified threshold level or within the specified target range isreached or maintained. For example, in certain embodiments fasting bloodammonia level may be measured after administration of the second dosage,and if the fasting blood ammonia level is at or above the specifiedthreshold level with respect to the ULN, a third dosage may beadministered that is greater than the second dosage. In certainembodiments, the specified threshold level is 1.5 times the ULN. Incertain embodiments, the specified target range is 1 to 1.5, between 1and 1.5, <1.5, or ≦1.5 times the ULN. In certain embodiments,administration of the nitrogen scavenging drug decreases the likelihoodof the subject experiencing an HE event.

Provided herein in certain embodiments are methods of evaluating theefficacy of a nitrogen scavenging drug for treatment of HE in a subjectin need thereof. In certain embodiments, these methods comprise (a)measuring a fasting blood ammonia level in a subject who has beenadministered a nitrogen scavenging drug and (b) comparing the fastingblood ammonia level to the ULN for blood ammonia, wherein a fastingblood ammonia level at or above a specified threshold level or above aspecified target range with respect to the ULN for blood ammoniaindicates the nitrogen scavenging drug has not been fully effective. Incertain embodiments, these methods comprise the additional step ofadministering an increased dosage of the nitrogen scavenging drug oradministering a second nitrogen scavenging drug in lieu of or inaddition to the original nitrogen scavenging drug. In certainembodiments, the specified threshold level is 1.5 times the ULN. Incertain embodiments, the specified target range is 1 to 1.5, between 1and 1.5, <1.5, or ≦1.5 times the ULN.

Provided herein in certain embodiments are methods of determiningwhether to administer a nitrogen scavenging drug for treatment of HE toa subject in need thereof comprising (a) measuring a fasting bloodammonia level and (b) comparing the fasting blood ammonia level to aspecified threshold level or a specified target range with respect tothe ULN for blood ammonia, wherein a fasting blood ammonia level at orabove the specified threshold level or above the specified target rangeindicates that a nitrogen scavenging drug should be administered to thesubject. In certain embodiments, these methods further compriseadministering the nitrogen scavenging drug. In certain embodiments, thespecified threshold level is 1.5 times the ULN. In certain embodiments,the specified target range is 1 to 1.5, between 1 and 1.5, <1.5, or ≦1.5times the ULN. In certain embodiments, administration of the nitrogenscavenging drug decreases the likelihood of the subject experiencing anHE event.

Provided herein in certain embodiments are methods of determiningwhether to administer a second nitrogen scavenging drug for treatment ofHE to a subject in need thereof who has previously been administered afirst nitrogen scavenging drug comprising (a) measuring a fasting bloodammonia level after administration of the first nitrogen scavenging drugand (b) comparing the fasting blood ammonia level to a specifiedthreshold level or specified target range with respect to the ULN forblood ammonia, wherein a fasting blood ammonia level at or above thespecified threshold level or above the specified target range indicatesthe need to administer a second nitrogen scavenging drug. In certainembodiments, these methods further comprise administering the secondnitrogen scavenging drug. In certain of these embodiments, the secondnitrogen scavenging drug is administered in lieu of the first nitrogenscavenging drug. In other embodiments, the second nitrogen scavengingdrug is administered in combination with the first nitrogen scavengingdrug, either sequentially or simultaneously. In certain embodiments, thespecified threshold level is 1.5 times the ULN. In certain embodiments,the specified target range is 1 to 1.5, between 1 and 1.5, <1.5, or ≦1.5times the ULN. In certain embodiments, administration of the secondnitrogen scavenging drug decreases the likelihood of the subjectexperiencing an HE event.

Provided herein in certain embodiments are methods of using fastingblood ammonia level to predict the likelihood and/or severity of futureHE events. In certain of these embodiments, a subject is classified asmore likely to experience an HE event or more likely to experience asevere HE event if the subject exhibits a fasting blood ammonia level ator above a specified threshold level or above a specified target rangewith respect to the ULN for blood ammonia. Similarly, in certainembodiments, a subject is classified as less likely to experience an HEevent or less likely to experience a severe HE event if the subjectexhibits a fasting blood ammonia level at or below a specified thresholdlevel or within a specified target range with respect to the ULN forblood ammonia. In certain embodiments, the specified threshold level is1.5 times the ULN. In certain embodiments, the specified target range is1 to 1.5, between 1 and 1.5, <1.5, or ≦1.5 times the ULN. In certainembodiments, a subject's risk of experiencing an HE event increases thehigher the fasting blood ammonia level rises above the specifiedthreshold level or specified target range. For example, where thespecified threshold level is 1.5, a subject with a fasting blood ammonialevel of 2.0 times the ULN for blood ammonia may be classified as morelikely to experience an HE event than a subject with a fasting bloodammonia level 1.6 times the ULN. Similarly, in certain embodiments, thelikely severity of a future HE event increases the higher the fastingblood ammonia level rises above the specified threshold value orspecified target range. For example, where the specified threshold levelis 1.5, a subject with a fasting blood ammonia level of 2.0 times theULN for blood ammonia may be expected to experience more severe HEevents than a subject with a fasting blood ammonia level 1.6 times theULN. In certain embodiments, these methods further comprise taking stepsto reduce the likelihood that the subject will experience an HE event,and in certain of these embodiments the methods comprise administering anitrogen scavenging drug or an increased dosage of a nitrogenscavenging. In certain of these embodiments, the nitrogen scavengingdrug is administered at a dosage sufficient to drop a subject's fastingblood ammonia level to or below a specified threshold level or to withina specified target range with respect to the ULN for blood ammonia, orto maintain the subject's fasting blood ammonia level at or below thespecified threshold level or within the specified target range for aspecific period of time.

The ULN for blood ammonia typically represents the highest level in therange of normal values, which may be influenced by a variety of factorssuch as the assay method, types of regents, standard reference samplesused, and specifications and calibration of equipment used to performthe measurement. In certain embodiments of the methods disclosed herein,the ULN for blood ammonia is determined for a subject individually. Inother embodiments, the ULN for blood ammonia may be based onmeasurements obtained across a set of subjects (e.g., healthy subjectsor subjects with HE). In certain embodiments, the ULN for blood ammoniamay represent a standard reference value disclosed in the art, such as amean ULN developed across a particular subset of subjects. In otherembodiments, the ULN for blood ammonia may represent a standardmeasurement that has been developed by a particular entity that performsblood draws and/or blood evaluations, such as a particular clinicallaboratory. In certain embodiments, the ULN is a standard referencevalue utilized by the same entity that measures the fasting bloodammonia level. In these embodiments, one skilled in the art willrecognize that the units of ammonia measurement may also vary from labto lab (e.g., μg/mL or μmoI/L), emphasizing the importance ofinterpreting the subject's ammonia levels relative to the ULN at thelaboratory in which the measurement was performed. In certainembodiments, the ULN for blood ammonia may be about 12 to 70 μmol/L. Incertain of these embodiments, the ULN for blood ammonia may be about 11to 64 μmol/L, 20 to 50 μmol/L, 30 to 40 μmol/L, 32 to 38 μmol/L, or 34to 36 μmol/L, and in certain of these embodiments the ULN for bloodammonia is about 35 μmol/L. In certain embodiments, the ULN for bloodammonia may be about 20 to 120 μg/dL. In certain of these embodiments,the ULN for blood ammonia may be about 50 to 65 μg/dL, 55 to 63 μg/dL,or 57 to 61 μg/dL, and in certain of these embodiments the ULN for bloodammonia is about 59 μg/dL.

A nitrogen scavenging drug as used herein refers to any drug thatdecreases blood nitrogen and/or ammonia levels. In certain embodiments,a nitrogen scavenging drug may remove nitrogen in the form of PAGN, andin certain of these embodiments the nitrogen scavenging drug may be anorally administrable drug that contains or is metabolized to PAA. Forexample, a nitrogen scavenging drug may be a PAA prodrug such as PBA orHPN-100, a pharmaceutically acceptable salt of PBA such as NaPBA, or apharmaceutically acceptable ester, acid, or derivative of a PAA prodrug.In other embodiments, a nitrogen scavenging drug may remove nitrogen viahippuric acid. In certain of these embodiments, a nitrogen scavengingdrug may be benzoic acid, a pharmaceutically acceptable salt of benzoicacid such as sodium benzoate, or a pharmaceutically acceptable ester,acid, or derivative of benzoic acid.

Increasing the dosage of a nitrogen scavenging drug may refer toincreasing the amount of drug per administration (e.g., an increase froma 3 mL dosage to a 6 mL dosage), increasing the number ofadministrations of the drug (e.g., an increase from once-a-day dosing totwice- or three-times-a-day), or any combination thereof.

In certain embodiments, a subject that has previously been administereda nitrogen scavenging drug has been administered the drug for a durationof time sufficient to reach steady state. Similarly, in those methodswhere fasting blood ammonia level is measured following a first, second,third, or subsequent dosage of nitrogen scavenging drug, the measurementmay be carried out after the drug has had sufficient time to reachsteady state at that dosage. For example, the subject may have beenadministered the drug over a period of about 2 to 7 days, 1 week to 2weeks, 2 weeks to 4 weeks, 4 weeks to 8 weeks, 8 weeks to 16 weeks, orlonger than 16 weeks.

In certain embodiments of the methods disclosed herein, the fastingperiod for obtaining a fasting blood ammonia level is overnight. Incertain embodiments, the fasting period is 4 hours or more, 5 hours ormore, 6 hours or more, 7 hours or more, 8 hours or more, 9 hours ormore, 10 hours or more, 11 hours or more, or 12 hours or more, and incertain embodiments the fasting period is 4 to 8 hours, 6 to 8 hours, or8 to 12 hours. During the fasting period, the subject preferably doesnot ingest any food. In certain embodiments, the subject may alsorefrain from ingesting certain non-food substances during the fastingperiod. For example, in certain embodiments the subject does not ingestany supplements and/or nitrogen scavenging drugs during the fastingperiod. In certain of these embodiments, the subject may nonethelessingest one or more drugs other than nitrogen scavenging drugs during thefasting period. In certain embodiments, the subject does not ingest anyhigh calorie liquids during the fasting period. In certain of theseembodiments, the subject does not ingest any liquids other than waterduring the fasting period. In other embodiments, the subject may ingestsmall amounts of low calorie beverages, such as tea, coffee, or dilutedjuices.

In certain embodiments of the methods disclosed herein, blood samplesused for measuring fasting blood ammonia levels and/or ULN bloodammonias are venous blood samples. In certain embodiments, a bloodsample is a plasma blood sample. Any methods known in the art may beused to obtain a plasma blood sample. For example, blood from a subjectmay be drawn into a tube containing heparin orethylenediaminetetraacetic acid (EDTA). In certain embodiments, thesample can be placed on ice and centrifuged to obtain plasma within 15minutes of collection, stored at 2 to 8° C. (36 to 46° F.) and analyzedwithin 3 hours of collection. In other embodiments, the blood plasmasample is snap frozen, stored at ≦−18° C. (≦0° F.) and analyzed at alater time. For example, the sample may be analyzed at 0 to 12 hours, 12to 24 hours, 24 to 48, 48 to 96 hours after freezing, or within anyother timeframe over which the sample has demonstrated stability. Incertain embodiments, blood samples are taken in a laboratory or hospitalsetting. In certain embodiments, a single fasting blood sample is usedto measure fasting blood ammonia level. However, in other embodiments,multiple fasting blood samples may be obtained. In certain embodiments,a subject's blood ammonia level may be monitored throughout the day.Further, in certain embodiments, the methods disclosed herein comprisean additional step of obtaining one or more blood samples from a subjectprior to or after measuring fasting blood ammonia level.

In certain embodiments, a blood sample is analyzed immediately aftercollection. In other embodiments, the blood sample is stored for someperiod between collection and analysis. In these embodiments, the samplemay be stored for less than 1 hour, 1 hour to 6 hours, 1 hour to 12hours, 1 hour to 24 hours, or 1 hour to 48 hours. In certain of theseembodiments, the blood sample is stored at a temperature between 0 to15° C., such as 2 to 8° C. In other embodiments, the blood sample isstored below 0° C. or below −18° C.

Measurement of ammonia levels in a fasting blood sample may be carriedout using any technique known in the art. For example, ammonia levelsmay be measured using a colorimetric reaction or an enzymatic reaction.In certain embodiments, a colorimetric reaction may involve the use ofbromophenol blue as an ammonia indicator. In these embodiments, ammoniamay react with bromophenol blue to yield a blue dye. In certainembodiments, an enzymatic reaction may involve glutamate dehydrogenasecatalyzing the reductive amination of 2-oxoglutarate with NH⁴⁺ and NADPHto form glutamate and NADP⁺. The formation of NADP⁺ formed is directlyproportional to the amount of ammonia present in the blood sample.Therefore, the concentration of ammonia is measured based on a decreasein absorbance.

As further set forth in the examples section below, the conversion ofPAA prodrugs to urinary PAGN was evaluated in subjects with HE. The meanpercent conversion of PAA prodrug to urinary PAGN was found to be 57%,with a 95% confident interval range of 52-63%. This is significantlylower than the conversion percentage reported previously for HE patientsin US Patent Publication No. 2010/0008859. US Patent Publication No.2010/0008859 reports an overall mean conversion percentage of 60-75% insubjects with nitrogen retention disorders generally, and a meanconversion percentage of approximately 75% in HE patients specifically.In certain embodiments, this mean conversion percentage of PAA prodrugto urinary PAGN may be used alone or in combination with fasting bloodammonia level measurements and/or dietary protein intake measurements totreat HE using a PAA prodrug, administer a PAA prodrug, determine aneffective dosage of a PAA prodrug, or evaluate or adjust the dosage of aPAA prodrug. In these embodiments, the mean percent conversion may be50-65% or a percentage falling within this range (e.g., about 52 to 63%,55 to 60%, 56 to 58%, 57%, etc.). In certain embodiments of the methodsdisclosed herein that take into account the mean percent conversion ofPAA prodrug to urinary PAGN, the effective dosage is calculated based ona target nitrogen output. In certain embodiments, urinary PAGN may bedetermined as a ratio of the concentration of urinary PAGN to urinarycreatinine.

In certain embodiments, methods are provided for calculating aneffective initial dosage of a PAA prodrug for a subject with HE bydetermining a target urinary PAGN output and calculating the effectiveinitial dosage based on the mean percent conversion of PAA prodrug tourinary PAGN as disclosed herein. In certain related embodiments,methods are provided for administering a PAA prodrug to a subject withHE or treating a subject having HE comprising (a) determining a targeturinary PAGN output, (b) calculating an effective initial dosage of PAAprodrug based on the mean percent conversion of PAA prodrug to urinaryPAGN, and (c) administering the effective initial dosage. In otherembodiments, methods are provided for determining an effective dosage ofa PAA prodrug for a subject with HE comprising (a) administering a firstdosage, (b) measuring urinary PAGN output, and (c) determining aneffective dosage based on the mean percent conversion of PAA prodrug tourinary PAGN as disclosed herein. In certain related embodiments,methods are provided for administering a PAA prodrug to a subject withHE comprising (a) administering a first dosage of PAA prodrug, (b)determining urinary PAGN output, (c) determining an effective dosage ofthe PAA prodrug based on the mean percent conversion of PAA prodrug tourinary PAGN, and (d) administering the effective dosage. In still otherembodiments, methods are provided for determining whether to adjust adosage of PAA prodrug based on the mean percent conversion of PAAprodrug to urinary PAGN as disclosed herein.

In certain embodiments, the percent conversion of PAA prodrug to urinaryPAGN as disclosed herein may be incorporated into the methods set forthherein for treating HE, optimally administering a nitrogen scavengingdrug for the treatment of HE, adjusting the dosage of a nitrogenscavenging drug for the treatment of HE, evaluating the efficacy of anitrogen scavenging drug for the treatment of HE, determining whether toadminister a nitrogen scavenging drug for the treatment of HE, orpredicting the likelihood or risk of an HE event based on fasting bloodammonia level. In certain of these embodiments, the percent conversionof PAA prodrug to urinary PAGN as disclosed herein may be used todetermine an effective initial dosage of PAA prodrug for treating HE,while the fasting ammonia level may be used to determine the efficacy ofthe initial dosage or to determine whether to adjust the dosage oradminister a second PAA prodrug. In other embodiments, the percentconversion of PAA prodrug to urinary PAGN as disclosed herein andfasting ammonia level may both be taken into account when determiningthe optimal first or subsequent dosage of a PAA prodrug, adjusting thedosage of a PAA prodrug, evaluating the efficacy of a PAA prodrug, ordetermining whether to administer a PAA prodrug for the treatment of HE.In certain of these embodiments, the methods include steps of measuringfasting ammonia level and measuring urinary PAGN.

One skilled in the art will recognize that a variety of other factorsmay be taken into consideration when determining the effective dosage ofa nitrogen scavenging drug. For example, factors such as diet (e.g.,protein intake) and endogenous waste nitrogen removal capacity (e.g.,urea synthesis capacity) may be considered.

Provided herein in certain embodiments are kits for carrying out themethods disclosed herein. In certain embodiments, kits are provided forevaluating the likelihood of a subject experiencing an HE event and fordetermining whether to administer a nitrogen scavenging drug or adjustthe dosage of a nitrogen scavenging drug for a subject. The kitsdisclosed herein may include one or more nitrogen scavenging drugsand/or one or more reagents (e.g., bromophenol blue) or enzymes (e.g.,glutamate dehydrogenase) to measure blood ammonia levels in a sample.The kit may additionally include other pigments, binders, surfactants,buffers, stabilizers, and/or chemicals necessary to obtain a bloodsample and to measure the ammonia level in the sample. In certainembodiments, the kits provided herein comprise instructions in atangible medium.

One of ordinary skill in the art will recognize that the variousembodiments described herein can be combined. For example, steps fromthe various methods of treatment disclosed herein may be combined inorder to achieve a satisfactory or improved level of treatment.

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention. It will be understood thatmany variations can be made in the procedures herein described whilestill remaining within the bounds of the present invention. It is theintention of the inventors that such variations are included within thescope of the invention.

EXAMPLES Example 1 Predicting Future HE Events Based on Fasting AmmoniaLevels

The relationship between HE events and fasting ammonia levels wasevaluated in subjects from the HALT-HE clinical trial (HPN-100-008,ClinicalTrials.gov identifier NCT00999167). To be eligible for HEanalysis, subject had to (1) be included in the HALT-HE (Part B) safetypopulation, (2) have at least one treatment with a study drug (eitherHPN-100 or placebo), and (3) have a quantifiable measurement of plasmaammonia from a blood specimen drawn at the visit and time point ofinterest. Of the 178 total subjects enrolled in the HALT-HE study andincluded in the safety population, 171 met the additional criteria.

Fasting ammonia levels were measured on day 1 (baseline) and at days 7and 14. Day 1 values were derived from the measurement closest to, butprior to, initiation of dosing. Day 7 and 14 values were obtained fromthe nominal visit day and time point (pre-dose or 4-hour). To accountfor differences in local laboratories used during the clinical trial,local measurements of ammonia were standardized to an upper limit ofnormal (ULN) of 35 μmol/L according to the following formula:

Ammonia_(std)=Ammonia_(local)*(35/ULN of Ammonia_(local)).

HE events were evaluated over 16 weeks. An HE event was defined as theoccurrence of either (1) a West Haven Grade 2 or above or (2) a WestHaven Grade 1 AND an asterixis grade increase of 1, if baseline WestHaven was 0.

To describe the relationship between plasma ammonia levels and HEevents, ammonia levels at the nominal times were initially grouped intofive categories as multiples of the standardized ULN (35 μmol/L): (1)[0-≦0.5], (2) [0.5-≦1.0], (3) [1.0-≦1.5], (4) [1.5-≦2.0], and (5)[>2.0].

Each subject had between zero and eight HE episodes over the course oftheir exposure to study drug. The relationship between ammonia at agiven nominal time and the number of HE events was modeled using anegative binomial model whereby the effect of study drug was consideredonly after controlling for ammonia level. Since not all subjects had thesame amount of time in the study (and thus did not have the sameopportunity for experiencing HE events), the model adjusted for (offsetfor) duration of treatment, measured in days and converted to weeks.Based on the model, the number of HE events was estimated for each levelof ammonia. By comparing each level of ammonia to the lowest level, thepattern of the relationship could not only be described but alsostatistically tested through a priori contrasts.

For purposes of prediction, the number of HE events for each subject wascategorized using two different outcome schemes: (1) a three categorygrouping (0 episodes, 1 episode, or 2 or more episodes) and (2) a twocategory grouping (0 episodes or 1 or more episodes). For the threecategory outcome scheme, the probability of having an outcome in one ofthe three categories based on ammonia level was modeled using orderedlogistic regression following a proportional odds model (McCullagh andNelder, Generalized Linear Models, 2nd ed. Boca Raton: Chapman and Hall,Ch. 5 (1989)). For the second scheme, probabilities of having an outcomein one of the two categories based on ammonia level were modeled usingbinary logistic regression. In each case, 95% confidence intervals ofprediction around the probabilities were derived using bootstrapresampling of the original dataset with 1000 iterations following thetechnique of Davison and Hinkley (Bootstrap Methods and TheirApplication, Cambridge Univ. Press, pp. 358-362 (1997)).

The relationship between HE events and ammonia levels at day 7(pre-dose), day 7 (four hours after dosing), day 14 (pre-dose), and day14 (four hours after dosing) for each of these groups is summarized inFIGS. 2-6. This analysis revealed no significant difference between thelowest two categories and between the highest two categories, so thecategories were collapsed into two different grouping schemes formodeling purposes: (1) a three category grouping ([0-1.0], [>1.0-1.5],and [>1.5]) and 2) a two category grouping ([0-1.5] and [>1.5]). Therelationship between HE events and ammonia levels at day 1, day 7(pre-dose), day 7 (four hours after dosing), day 14 (pre-dose), and day14 (four hours after dosing) for each of these groups is summarized inTables 1−5, respectively. In each table, the first three rows for eachtreatment group show results for the three category model ([0-1.0],[>1.0-1.5], and [>1.5]), while the next two rows show results for thetwo category model ([0-1.5] and [>1.5]). Note that, based on thestatistical methodology, estimation of HE events for input category >1.5in a two category model is not necessarily equal to the result for thethree category model.

TABLE 1 Count of HE events over 16 weeks by level of pre-dose ammonia onday 1: Plasma ammonia No. of per-subject HE Events p-value levelEstimated* Estimated* p-value p-value drug × Treatment (as multiple overover vs. lowest main effect ammonia Group of ULN) N Min Max 16 weeks 52weeks level of drug interaction HPN-100 [0-1.0] 37 0 2 0.25 0.80 . . .[>1.0-1.5]    23 0 5 0.61 1.98 0.287 . . [>1.5] 26 0 4 1.35 4.37 0.031 .. [0-1.5] 60 0 5 0.39 1.26 . . . [>1.5] 26 0 4 1.36 4.41 0.071 . .Placebo [0-1.0] 34 0 2 0.37 1.21 . . . [>1.0-1.5]    22 0 5 0.95 3.080.119 . . [>1.5] 29 0 8 2.20 7.14 <.001 . . [0-1.5] 56 0 5 0.59 1.90 . .. [>1.5] 29 0 8 2.21 7.19 0.003 . . TOTAL [0-1.0] 71 0 2 0.30 0.99 .0.132 0.995 [>1.0-1.5]    45 0 5 0.78 2.55 0.054 . . [>1.5] 55 0 8 1.815.89 <.001 . . [0-1.5] 116 0 5 0.48 1.57 . . . [>1.5] 55 0 8 1.83 5.94<.001 . .

TABLE 2 Count of HE events over 16 weeks by level of pre-dose ammonia onday 7: No. of per-subject HE Events p-value Plasma Ammonia Estimated*Estimated* p-value p-value drug × Treatment (as multiple over over vs.lowest main effect ammonia Group of ULN) N Min Max 16 weeks 52 weekslevel of drug interaction HPN-100 [0-1.0] 43 0 2 0.29 0.95 . . .[>1.0-1.5]    17 0 5 0.90 2.92 0.174 . . [>1.5] 18 0 4 1.38 4.50 0.046 .. [0-1.5] 60 0 5 0.45 1.47 . . . [>1.5] 18 0 4 1.40 4.56 0.134 . .Placebo [0-1.0] 33 0 1 0.30 0.97 . . . [>1.0-1.5]    19 0 2 0.23 0.730.699 . . [>1.5] 26 0 8 2.53 8.23 <.001 . . [0-1.5] 52 0 2 0.27 0.88 . .. [>1.5] 26 0 8 2.53 8.22 <.001 . . TOTAL [0-1.0] 76 0 2 0.29 0.94 .0.920 0.124 [>1.0-1.5]    36 0 5 0.47 1.53 0.348 . . [>1.5] 44 0 8 2.106.84 <.001 . . [0-1.5] 112 0 5 0.35 1.13 . . . [>1.5] 44 0 8 2.11 6.85<.001 . .

TABLE 3 Count of HE events over 16 weeks by level of ammonia on day 7,four hours post-dose: No. of per-subject HE Events p-value PlasmaAmmonia Estimated* Estimated* p-value p-value drug × Treatment (asmultiple over over vs. lowest main effect ammonia Group of ULN) N MinMax 16 weeks 52 weeks level of drug interaction HPN-100 [0-1.0] 33 0 20.16 0.51 . . . [>1.0-1.5]    20 0 5 0.88 2.85 0.048 . . [>1.5] 23 0 41.16 3.77 0.016 . . [0-1.5] 53 0 5 0.41 1.34 . . . [>1.5] 23 0 4 1.183.85 0.145 . . Placebo [0-1.0] 36 0 4 0.36 1.17 . . . [>1.0-1.5]    12 02 0.36 1.16 0.999 . . [>1.5] 30 0 8 2.19 7.13 <.001 . . [0-1.5] 48 0 40.36 1.16 . . . [>1.5] 30 0 8 2.19 7.13 <.001 . . TOTAL [0-1.0] 69 0 40.27 0.87 . 0.275 0.249 [>1.0-1.5]    32 0 5 0.63 2.05 0.111 . . [>1.5]53 0 8 1.76 5.72 <.001 . . [0-1.5] 101 0 5 0.38 1.23 . . . [>1.5] 53 0 81.77 5.74 <.001 . .

TABLE 4 Count of HE events over 16 weeks by level of ammonia on day 14:No. of per-subject HE Events p-value Plasma Ammonia Estimated*Estimated* p-value p-value drug × Treatment (as multiple over over vs.lowest main effect ammonia Group of ULN) N Min Max 16 weeks 52 weekslevel of drug interaction HPN-100 [0-1.0] 45 0 5 0.36 1.18 . . .[>1.0-1.5]    14 0 2 0.40 1.29 0.923 . . [>1.5] 19 0 4 1.29 4.21 0.106 .. [0-1.5] 59 0 5 0.37 1.21 . . . [>1.5] 19 0 4 1.29 4.21 0.096 . .Placebo [0-1.0] 31 0 4 0.53 1.73 . . . [>1.0-1.5]    16 0 2 0.20 0.650.230 . . [>1.5] 27 0 8 2.08 6.76 0.013 . . [0-1.5] 47 0 4 0.41 1.32 . .. [>1.5] 27 0 8 2.08 6.77 0.001 . . TOTAL [0-1.0] 76 0 5 0.43 1.41 .0.428 0.579 [>1.0-1.5]    30 0 2 0.28 0.92 0.474 . . [>1.5] 46 0 8 1.775.74 0.002 . . [0-1.5] 106 0 5 0.39 1.26 . . . [>1.5] 46 0 8 1.77 5.74<.001 . .

TABLE 5 Count of HE events over 16 weeks by level of ammonia on day 14,four hours post-dose: No. of per-subject HE Events p-value PlasmaAmmonia Estimated* Estimated* p-value p-value drug × Treatment (asmultiple over over vs. lowest main effect ammonia Group of ULN) N MinMax 16 weeks 52 weeks level of drug interaction HPN-100 [0-1.0] 35 0 40.28 0.92 . . . [>1.0-1.5]    22 0 2 0.41 1.33 0.688 . . [>1.5] 17 0 40.96 3.13 0.210 . . [0-1.5] 57 0 4 0.33 1.09 . . . [>1.5] 17 0 4 0.973.14 0.236 . . Placebo [0-1.0] 35 0 1 0.20 0.65 . . . [>1.0-1.5]    7 01 0.47 1.52 0.360 . . [>1.5] 34 0 8 1.97 6.40 <.001 . . [0-1.5] 42 0 10.25 0.81 . . . [>1.5] 34 0 8 1.97 6.42 <.001 . . TOTAL [0-1.0] 70 0 40.24 0.77 . 0.474 0.493 [>1.0-1.5]    29 0 2 0.41 1.35 0.346 . . [>1.5]51 0 8 1.66 5.39 <.001 . . [0-1.5] 99 0 4 0.29 0.95 . . . [>1.5] 51 0 81.66 5.41 <.001 . .

As shown in these results, fasting ammonia levels were correlated withsubsequent HE events over the 16 week exposure period. For example,subjects with a pre-dose ammonia level on day 1 that was less than orequal to the ULN (i.e., 0-1.0) experienced an estimated 0.30 events over16 weeks. The number of events increased to 0.78 for subjects withpre-dose ammonia level of >1.0 to 1.5 ULN, and to 1.81 for subjects withammonia levels greater than 1.5 ULN. The same pattern was observed ineach study drug treatment arm.

The number of events in the highest level ammonia group (>1.5 ULN) wassignificantly greater than the number of events in the lowest levelgroup (0 to 1.0 ULN) (p<0.001), and the number of events in the middlelevel ammonia group (>1.0 to 1.5 ULN) was marginally greater than thenumber of events in the lowest level group (p=0.054). These results showthat the risk of an HE event does not increase linearly with increasingammonia level. Instead, there appears to be a step up in the risk forbaseline ammonia levels greater than 1.5 times the ULN. This trendappears to be evident regardless of which visit and time the ammonia wasdrawn.

The proportional odds of experiencing an HE for various ammonia levelsat day 1, day 7 (pre-dose), day 7 (four hours after dosing), day 14(pre-dose), and day 14 (four hours after dosing) are summarized inTables 6−10, respectively. Levels 1, 2, and 4 in each table show theresults for the three category model ([0-1.0], [>1.0-1.5], and [>1.5]),while levels 3 and 4 show the results for the two category model([0-1.5] and [>1.5]).

TABLE 6 Probably of HE events over 16 weeks as predicted by level ofpre-dose ammonia on day 1: Plasma ammonia as predictor p-value PlasmaAmmonia Prob. of having this number of HE events (95% CI) vs. next (asmultiple 2 or more 1 or more lower Level of ULN) 0 events 1 event eventsevents level 1 [0-1.0] 0.812 0.114 0.074 0.197 (0.722, 0.896) (0.059,0.180) (0.037, 0.113) (0.107, 0.297) 2 [>1.0-1.5]    0.758 0.143 0.0990.244 0.547 (0.627, 0.883) (0.070, 0.220) (0.040, 0.174) (0.116, 0.376)3 [0-1.5] 0.792 0.125 0.083 0.216 (0.721, 0.865) (0.071, 0.181) (0.049,0.120) (0.138, 0.289) 4 [>1.5] 0.529 0.236 0.235 0.436 0.003 (0.386,0.672) (0.161, 0.319) (0.124, 0.359) (0.306, 0.574)

TABLE 7 Probably of HE events over 16 weeks as predicted by level ofpre-dose ammonia on day 7: Plasma ammonia as predictor p-value PlasmaAmmonia Prob. of having this number of HE events (95% CI) vs. next (asmultiple 2 or more 1 or more lower Level of ULN) 0 events 1 event eventsevents level 1 [0-1.0] 0.812 0.118 0.070 0.197 (0.732, 0.889) (0.064,0.182) (0.038, 0.106) (0.114, 0.285) 2 [>1.0-1.5]    0.830 0.108 0.0630.167 0.698 (0.692, 0.944) (0.040, 0.184) (0.017, 0.132) (0.055, 0.293)3 [0-1.5] 0.817 0.115 0.068 0.188 (0.750, 0.882) (0.069, 0.168) (0.039,0.104) (0.120, 0.257) 4 [>1.5] 0.466 0.262 0.272 0.500 <0.001 (0.318,0.617) (0.176, 0.351) (0.148, 0.406) (0.356, 0.646)

TABLE 8 Probably of HE events over 16 weeks as predicted by level ofammonia on day 7, four hours post-dose: Plasma ammonia as predictorp-value Plasma Ammonia Prob. of having this number of HE events (95% CI)vs. next (as multiple 2 or more 1 or more lower Level of ULN) 0 events 1event events events level 1 [0-1.0] 0.846 0.091 0.063 0.159 (0.769,0.921) (0.044, 0.151) (0.031, 0.098) (0.081, 0.246) 2 [>1.0-1.5]   0.754 0.138 0.108 0.250 0.282 (0.611, 0.900) (0.059, 0.218) (0.038,0.198) (0.100, 0.403) 3 [0-1.5] 0.817 0.106 0.077 0.188 (0.746, 0.886)(0.060, 0.162) (0.043, 0.115) (0.117, 0.263) 4 [>1.5] 0.540 0.220 0.2400.434 0.001 (0.402, 0.675) (0.147, 0.301) (0.131, 0.357) (0.305, 0.566)

TABLE 9 Probably of HE events over 16 weeks as predicted by level ofpre-dose ammonia on day 14: Plasma ammonia as predictor p-value PlasmaAmmonia Prob. of having this number of HE events (95% CI) vs. next (asmultiple 2 or more 1 or more lower Level of ULN) 0 events 1 event eventsevents level 1 [0-1.0] 0.812 0.104 0.084 0.197 (0.731, 0.888) (0.055,0.163) (0.047, 0.126) (0.116, 0.287) 2 [>1.0-1.5]    0.834 0.093 0.0730.167 0.716 (0.699, 0.945) (0.026, 0.169) (0.018, 0.144) (0.054, 0.309)3 [0-1.5] 0.818 0.101 0.081 0.189 (0.747, 0.884) (0.055, 0.154) (0.049,0.117) (0.118, 0.263) 4 [>1.5] 0.525 0.211 0.264 0.435 0.002 (0.377,0.678) (0.136, 0.287) (0.134, 0.398) (0.298, 0.571)

TABLE 10 Probably of HE events over 16 weeks as predicted by level ofammonia on day 14, four hours post-dose: Plasma ammonia as predictorp-value Plasma Ammonia Prob. of having this number of HE events (95% CI)vs. next (as multiple 2 or more 1 or more lower Level of ULN) 0 events 1event events events level 1 [0-1.0] 0.874 0.075 0.051 0.129 (0.801,0.941) (0.032, 0.129) (0.022, 0.086) (0.061, 0.203) 2 [>1.0-1.5]   0.738 0.145 0.117 0.276 0.083 (0.596, 0.880) (0.068, 0.234) (0.043,0.203) (0.121, 0.444) 3 [0-1.5] 0.834 0.097 0.070 0.172 (0.764, 0.896)(0.053, 0.154) (0.039, 0.108) (0.106, 0.245) 4 [>1.5] 0.561 0.213 0.2260.412 0.002 (0.425, 0.699) (0.138, 0.293) (0.120, 0.346) (0.285, 0.535)

Among subjects whose ammonia levels were normal pre-dose on day 1 (i.e.,0 to 1.0 ULN), 81% are expected to have no HE events within 16 weeks and7% are expected to have two or more HE events (Table 6). Among subjectwith day 1 pre-dose ammonia levels greater than 1.5 ULN, on the otherhand, only 52% are expected to have zero HE events within 16 weeks and23% are expected to have two or more HE events. As illustrated in thebaseline and day 7 results summarized in FIGS. 7-10, this pattern wasconsistent regardless of whether blood samples were drawn prior to thestudy or during the study.

Example 2 Conversion of PAA Prodrug to Urinary PAGN

US Patent Publication No. 2010/0008859 discloses that urinary PAGNlevels correlate more closely to PBA prodrug dosage than plasma PAA,PBA, or PAGN levels, and further discloses that PBA prodrugs areconverted to urinary PAGN with a mean efficiency of 60 to 75%. Thisconversion percentage is based on data obtained from subjects with UCDand HE. For HE subjects, PAA metabolism was evaluated by administeringHPN-100 to 32 subjects with hepatic impairment with cirrhosis. Thesubjects were broken into four groups of 8 subjects each: hepaticimpairment with cirrhosis and Child-Pugh scores of A, B, or C, and acontrol group of healthy adults with normal hepatic function. The meanpercent conversion of HPN-100 to urinary PAGN in these four patientgroups was of 79.6% (SD=30.5), 58.2% (SD=29.2), 85.0% (SD=65.1), and68.6% (SD=21.9), respectively. The overall mean percent conversion forthe four subject groups was about 75%.

PAA prodrug conversion has now been evaluated in additional HE subjects.These results unexpectedly show that the percent conversion of PAAprodrug to urinary PAGN is lower than disclosed in Patent PublicationNo. 2010/0008859.

As disclosed herein, additional trials have been performed to evaluatethe conversion of PAA to urinary PAGN in subjects with HE. Urinary PAGNoutput was measured in 130 HE patients during steady state dosing witheither NaPBA or an equivalent dose of HPN-100. As summarized in Table11, the mean percent conversion of PAA prodrug to urinary PAGN in HEpatients was about 57%, with a 95% confidence interval range of 52-63%.Results were consistent for both sodium PBA and HPN-100, indicating thatsimilar results would be expected for all PAA prodrugs. These resultssuggest that the percent conversion of PAA prodrug to urinary PAGN insubjects with HE is significantly lower than previously reported in USPatent Publication No. 2010/0008859.

TABLE 11 Mean percent conversion of PAA prodrugs to urinary PAGN insubjects with HE: N 130 Mean % conversion    57% 95% CI range 52-62%

As stated above, the foregoing is merely intended to illustrate variousembodiments of the present invention. The specific modificationsdiscussed above are not to be construed as limitations on the scope ofthe invention. It will be apparent to one skilled in the art thatvarious equivalents, changes, and modifications may be made withoutdeparting from the scope of the invention, and it is understood thatsuch equivalent embodiments are to be included herein. All referencescited herein are incorporated by reference as if fully set forth herein.

REFERENCES

-   1. Brusilow Science 207:659 (1980)-   2. Brusilow Pediatr Res 29:147 (1991)-   3. Diaz Mol Genet Metab 102:276 (2011)-   4. Ghabril Gastroenterology 142:S918 (2012)-   5. Lee Mol Genet Metab 100:221 (2010)-   6. Lichter-Konecki Mol Genet Metab 103:323 (2011)-   7. McGuire Hepatology 51:2077 (2010)-   8. Rockey Hepatology 56:248 (2012)

1.-12. (canceled)
 13. A method of treating hepatic encephalopathy (HE)in a subject comprising: (a) measuring a fasting blood ammonia level;(b) comparing the fasting blood ammonia level to the upper limit ofnormal for blood ammonia; and (c) administering sodium phenylbutyrate tothe subject if the fasting blood ammonia level is greater than 1.5 timesthe upper limit of normal for blood ammonia.
 14. The method of claim 13,wherein the subject has previously been administered a first dosage ofsodium phenylbutyrate.
 15. The method of claim 14, wherein the dosage ofsodium phenylbutyrate administered in step (c) is greater than the firstdosage.
 16. A method of optimizing the dosage of a nitrogen scavengingdrug for the treatment of hepatic encephalopathy (HE) comprising: (a)administering a first dosage of sodium phenylbutyrate; (b) measuring afasting blood ammonia level; (c) comparing the fasting blood ammonialevel to the upper limit of normal for blood ammonia to determinewhether to increase the dosage of sodium phenylbutyrate, wherein thedosage needs to be increased if the fasting blood ammonia level isgreater than 1.5 times the upper limit of normal for blood ammonia; and(d) administering a second dosage of sodium phenylbutyrate based on thedetermination in (c).
 17. The method of claim 13, further comprising astep of determining the upper limit of normal for blood ammonia for thesubject.
 18. The method of claim 16, further comprising a step ofdetermining the upper limit of normal for blood ammonia for the subject.19. The method of claim 13, wherein the upper limit of normal bloodammonia is 35 μmol/L.
 20. The method of claim 16, wherein the upperlimit of normal blood ammonia is 35 μmol/L.